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DE10240035A1 - Biogenic polyester particles of a defined size with functionalized surfaces: manufacturing processes and pharmaceutical preparations containing them - Google Patents

Biogenic polyester particles of a defined size with functionalized surfaces: manufacturing processes and pharmaceutical preparations containing them Download PDF

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DE10240035A1
DE10240035A1 DE10240035A DE10240035A DE10240035A1 DE 10240035 A1 DE10240035 A1 DE 10240035A1 DE 10240035 A DE10240035 A DE 10240035A DE 10240035 A DE10240035 A DE 10240035A DE 10240035 A1 DE10240035 A1 DE 10240035A1
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polyester
proteins
particles
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Priority to DE10240035A priority Critical patent/DE10240035A1/en
Priority to CA2497255A priority patent/CA2497255C/en
Priority to ES03747816T priority patent/ES2360617T3/en
Priority to EP10181200A priority patent/EP2302064A1/en
Priority to DK03747816.1T priority patent/DK1539976T3/en
Priority to DE50313457T priority patent/DE50313457D1/en
Priority to AT03747816T priority patent/ATE497543T1/en
Priority to AU2003266922A priority patent/AU2003266922B2/en
Priority to PCT/DE2003/002799 priority patent/WO2004020623A2/en
Priority to SI200331989T priority patent/SI1539976T1/en
Priority to EP03747816A priority patent/EP1539976B1/en
Priority to SG200701490-5A priority patent/SG153663A1/en
Priority to NZ569097A priority patent/NZ569097A/en
Priority to JP2004531694A priority patent/JP5160729B2/en
Priority to PT03747816T priority patent/PT1539976E/en
Priority to US10/525,955 priority patent/US7622277B2/en
Publication of DE10240035A1 publication Critical patent/DE10240035A1/en
Priority to AU2009210395A priority patent/AU2009210395A1/en
Priority to US12/588,227 priority patent/US20100086972A1/en
Priority to CY20111100420T priority patent/CY1113685T1/en
Priority to JP2012152732A priority patent/JP2012249634A/en
Priority to US14/143,725 priority patent/US20140206759A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
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  • Biochemistry (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Neurosurgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Polyesters Or Polycarbonates (AREA)

Abstract

A carrier and localization system for active or functional molecules (A) comprising particles of biodegradable polyester (I) having a single or multiple functionalized surface layer of proteins covalently or non-covalently bonded to (I) and/or amphiphilic molecules non-covalently bonded to (I), is new.

Description

Die Polyester-Nanopartikel (10-100 nm), -Submikropartikel (100-900 nm) und – Mikropartikel (1-3 μm) werden aus Wildtyp-Mikroorganismen (Eu- und Archaebakterien z. B. Ralstonia eutropha, Alcaligenes latus, Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas fluorescens, Halobiforma haloterrestris) oder gentechnisch veränderten Mikroorganismen der erwähnten Gruppen ( z. B. Escherichia coli, Pseudomonas fragi, Pseudomonaden, Ralstonia eutropha), die in der Lage sind Polyester aus Hydroxyfettsäuren und/oder Mercaptofettsäuren und/oder anderer Bausteine, die von Polyester-synthetisierenden Enzymen polymerisiert werden, zu synthetisieren. Unter anderen Bausteinen sind sämtliche Bausteine, die durch die Polyester-synthetisierende Enzyme zu einem Polyester polymerisiert werden können, zusammengefasst.The polyester nanoparticles (10-100 nm), -submicroparticles (100-900 nm) and - microparticles (1-3 μm) from wild-type microorganisms (eu and archaebacteria e.g. Ralstonia eutropha, Alcaligenes latus, Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas fluorescens, Halobiforma haloterrestris) or genetically modified Microorganisms of the mentioned Groups (e.g. Escherichia coli, Pseudomonas fragi, Pseudomonads, Ralstonia eutropha), which are capable of hydroxy fatty acids and / or polyesters Mercaptofettsäuren and / or other building blocks made from polyester-synthesizing Enzymes are polymerized to synthesize. Among other building blocks are all Building blocks created by the polyester-synthesizing enzymes Polyester can be polymerized summarized.

Entsprechende Mikroorganismen werden in geeigneten Minimal- oder Komplex-Medien mit geeigneten Vorstufensubstraten (Fettsäuren, Mercaptosäuren und Vorstufensubstrate, die zur einer Bildung von Polyester-Granula führen) kultiviert und in der spätstationären Wachstumsphase geerntet. Durch Wahl der Kultivierungsbedingungen (Vorstufensubstrate, Sequentielle Vorstufensubstratfütterung), Mikroorganismen (Wildtypen, Mutanten und gentechnisch veränderte Mikroorganismen) und der Stoffwechselflusssteuerung (Inhibitoren von Stoffwechselflüssen, Etablierung von Stoffwechselwegen durch gentechnische Veränderung) wird das mögliche Spektrum an unterschiedlichen Polyesterkernen erfasst. Der Zellaufschluss erfolgt entweder durch enzymatische oder durch mechanische Verfahren.Corresponding microorganisms are in suitable minimal or complex media with suitable prepress substrates (Fatty acids, mercapto and precursor substrates used to form polyester granules to lead) cultivated and in the late inpatient growth phase harvested. By choosing the cultivation conditions (precursor substrates, Sequential pre-stage substrate feeding), Microorganisms (wild types, mutants and genetically modified microorganisms) and metabolic flow control (inhibitors of metabolic flows, establishment of metabolic pathways due to genetic modification) becomes the possible spectrum recorded on different polyester cores. The cell disruption is carried out either by enzymatic or by mechanical processes.

Die Größe der Granula wird in vivo durch Verwendung einer phaP-negativen R. eutropha-Mutanten (knock out-Mutanten, York et al., 2001) und kontrollierter Expression von Phasin-Genen (ausgehend vom bad-Promotor mit Arabinose als Induktor) oder durch Verwendung von rekombinanten Mikroorganismen, welche Polyester synthetisieren können, und durch gleichzeitige kontrollierte Expression von Phasin-Genen kontrolliert. Hier werden alle mikrobiellen Systeme genutzt, die eine entsprechende Kontrolle der Granula-Größe ermöglichen. Die Phasin-Kopienzahl verhält sich grundsätzlich reziprok zur Größe der Granula (Wieczorek et al., 1995). Durch kontrollierte Erhöhung der Kopienzahl von Phasinen wird eine Abnahme des durchschnittlichen Granulum-Durchmessers bei gleichzeitiger Zunahme der Anzahl der Granula erreicht. Weiterhin wird die Größe der Granula durch die Kopienzahl der PHA-Synthase (PHA = Polyhydroxyalkanoat) und die Dauer der Verfügbarkeit der PHA-Synthase-Substrate kontrolliert. Durch Antisensetechnologie und/oder genetische Regulation und/oder Verfügbarkeit von Vorstufensubstraten wird die Bereitstellung von PHA-Synthase-Substraten kontrolliert. Weiterhin werden Granula unterschiedlicher Größe durch Gelfiltrationschromatographie und/oder Ultrafiltration und/oder Dichte-gradientenzentrifugation bezüglich der Göße fraktioniert (⌀ 10-500 nm).The size of the granules is in vivo by using a phaP-negative R. eutropha mutant (knock out mutants, York et al., 2001) and controlled expression of Phasin genes (starting from the bad promoter with arabinose as inducer) or by using recombinant microorganisms which are polyester can synthesize, and controlled by simultaneous controlled expression of phasin genes. All microbial systems are used here that have a corresponding Allow control of granule size. The phasin copy number behaves yourself basically reciprocal to the size of the granules (Wieczorek et al., 1995). By controlled increase in Copy number of phasins will decrease the average granule diameter reached while increasing the number of granules. Farther becomes the size of the granules by the copy number of the PHA synthase (PHA = polyhydroxyalkanoate) and the duration of availability controlled the PHA synthase substrates. Through antisense technology and / or genetic regulation and / or availability of precursor substrates the supply of PHA synthase substrates is controlled. Furthermore, granules of different sizes are obtained by gel filtration chromatography and / or ultrafiltration and / or density gradient centrifugation regarding the Göss fractionated (⌀ 10-500 nm).

Unterschiedliche PHA-Synthasen oder Lipasen oder PHA-Depolymerasen werden mittels His-tag-Fusion und Ni-NTA-Agarose-Affinitätchromatographie gereinigt. Die Polyester-synthetisierenden Enzyme und Coenzym A Thioster-bildende Enzyme (z. B. Acyl-CoA-Synthetasen) werden für die in vitro Synthese ausgehend von geeigneten Substraten (Substratgemischen, sequentielle Verfügbarkeit von Substraten), die zu Polyester-Vorstufen umgesetzt werden können, eingesetzt. Durch Erhöhung des Verhältnisses Polyester-synthetisierender Enzyme zur Substratkonzentration wird zu dem das durchschnittliche Molekulargewicht des Polyesters verringert (Sim et al., 1997). Die Oberfläche der Granula wird nach Bedarf mit Phospholipiden und/oder Etherlipiden und/oder Proteinen versehen werden, indem diese Komponenten dem in vitro Synthese-Ansatz in geeigneten Konzentration hinzugegeben werden. Durch gleichzeitige Verwendung von PHA-Synthasen mit unterschiedlicher Substratspezifität in Gegenwart unterschiedlicher Substrate werden Granula mit einem Polymer-Blend als Kernstruktur gewonnen. Bei der in vitro Polyester-Synthese werden ebenso nach Bedarf therapeutisch wirksame Substanzen hinzugegeben, die dann im Polyester-Kern vorliegen.Different PHA synthases or Lipases or PHA depolymerases are made using His-Tag fusion and Ni-NTA-agarose affinity chromatography cleaned. The polyester-synthesizing enzymes and coenzyme A Thioster-forming enzymes (e.g. acyl-CoA synthetases) are used for the in vitro synthesis starting from suitable substrates (substrate mixtures, sequential availability of substrates) that can be converted into polyester precursors. By increasing of the relationship Polyester-synthesizing enzymes for substrate concentration to which the average molecular weight of the polyester is reduced (Sim et al., 1997). The surface the granules are filled with phospholipids and / or ether lipids as required and / or proteins can be provided by these components added in vitro synthesis approach in suitable concentration become. By simultaneous use of PHA synthases with different substrate specificity in the presence Different substrates are granules with a polymer blend won as core structure. The same applies to in vitro polyester synthesis if necessary, therapeutically active substances are added, the then present in the polyester core.

Die unterschiedlichen Granula werden durch Standardverfahren aus den Bakterien bzw. dem in vitro Syntheseansatz gewonnen und anschließend durch Gelfiltrationchromatographie und/oder Ultrafiltration in 5 mM Phosphatpuffer pH 7,5 und/oder Dichtegradientenzentrifugation gemäß der Größe fraktioniert.The different granules are by standard methods from the bacteria or the in vitro synthesis approach obtained and then by gel filtration chromatography and / or ultrafiltration in 5 mM phosphate buffer pH 7.5 and / or Density gradient centrifugation fractionated according to size.

Granula, die aus Mikroorganismen oder aus der in vitro Synthese gewonnen wurden, werden bezüglich der Oberfläche modifiziert. Durch Einsatz von Phospholipasen und/oder Aceton-Extraktion und/oder Detergenzien werden Phospholipide von der Granulum-Obertläche entfernt und können durch Proteine und/oder andere amphiphile Moleküle ersetzt werden.Granules made up of microorganisms or obtained from the in vitro synthesis, with respect to the surface modified. By using phospholipases and / or acetone extraction and / or Detergents are removed from the granule surface by phospholipids and can be replaced by proteins and / or other amphiphilic molecules.

Funktionalisierung der Polyester-Partikel-Oberflächefunctionalization the polyester particle surface

Kovalent gebundene ProteineCovalently bound proteins

Im Rahmen der Erfindung getätigte Untersuchungen zeigen das die PHA-Synthase weder durch Behandlung mit denaturierenden Reagenzien (SDS, Harnstoff, Guanidiumhydrochlorid, DTT) noch durch Verwendung azider Bedingungen von dem Polyester-Kern gelöst werden können, was auf eine bestehende kovalente Verknüpfung mit dem Polyestermolekül anzeigt. Der N-terminale Abschnitt der PHA-Synthasen (N-Terminus bis zum Beginn der konservierten α/β-Hydrolase-Domäne) ist äußerst variable und wird durch gentechnische Methoden durch Funktionsproteine ersetzt werden, wobei die PHA-Synthase-Aktivität und Synthese von Granula erhalten bleibt (siehe auch Rehm et al., 2002). Folglich wird eine Funktionalisierung der Oberfläche erreicht. Ein Gemisch unterschiedlicher Fusionsproteine wird bei Bedarf gleichzeitig appliziert werden, so dass eine Multifunktionalisierung der Granulum-Oberfläche erzeugt wird. Die Applikation dieser Fusionsproteine erfolgt in vitro durch Zugabe der gereinigten Fusionsproteine zum Synthese-Ansatz bzw. in vivo durch Expression der Gene in dem entsprechendem Mikroorganismus, die für die Fusionsproteine kodieren.Investigations carried out within the scope of the invention show that the PHA synthase can neither be removed from the polyester core by treatment with denaturing reagents (SDS, urea, guanidium hydrochloride, DTT) nor by using acidic conditions, which indicates an existing covalent linkage with the polyester molecule displays. The N-terminal section of the PHA synthases (N-terminus up to the beginning of the conserved α / β-hydrolase domain) is extreme variable and will be replaced by genetic engineering methods with functional proteins, whereby the PHA synthase activity and synthesis of granules are retained (see also Rehm et al., 2002). Functionalization of the surface is consequently achieved. A mixture of different fusion proteins will be applied simultaneously if necessary, so that a multifunctionalization of the granule surface is created. These fusion proteins are applied in vitro by adding the purified fusion proteins to the synthesis approach or in vivo by expressing the genes in the corresponding microorganism which code for the fusion proteins.

Nicht-kovalent gebundene ProteineNon-covalently bound proteins

Der C-Terminus (> Ala 141 gemäß des Phasins PhaP aus R. eutropha) der Phasine ist hydrophil und wird durch Funktionsproteine ersetzt, ohne eine Verankerung in der Granulum-Oberfläche zu verhindern. Diese Verankerung beruht auf hydrophoben Wechselwirkungen und ist reversibel (Hanley et al., 1999). Ein Gemisch unterschiedlicher Fusionsproteine wird bei Bedarf gleichzeitig appliziert, so dass eine Multifunktionalisierung der Granulum-Oberfläche erzeugt wird.The C-terminus (> Ala 141 according to the phasin PhaP from R. eutropha) the phasin is hydrophilic and is replaced by functional proteins, without preventing anchoring in the granule surface. This anchoring is based on hydrophobic interactions and is reversible (Hanley et al., 1999). A mixture of different fusion proteins will if necessary applied simultaneously, so that a multifunctionalization the granule surface is produced.

Der C-Terminus (> Aminosäurerest 180 gemäß der intrazellulären PHA-Depolymerase von R. eutropha) der intrazellulären PHA-Depolymerasen vermittelt die Bindung des Enzyms an den Polyester-Kern der Granula (siehe auch Saegusa et al., 2001). Dieser C-terminate Abschnitt der intrazellulären PHA-Depolymerasen wird durch gentechnische Methoden an Funktionsmoleküle fusioniert und ermöglicht somit eine Funktionalisierung der Granulum-Oberfläche. Ein Gemisch unterschiedlicher Fusionsproteine wird bei Bedarf gleichzeitig appliziert, so dass eine Multifunktionalisierung der Granulum-Oberfläche erzeugt wird.The C-terminus (> amino acid residue 180 according to the intracellular PHA depolymerase from R. eutropha) of the intracellular PHA depolymerases mediate the binding of the enzyme to the polyester core the granules (see also Saegusa et al., 2001). This C-terminate Section of the intracellular PHA depolymerases are fused to functional molecules using genetic engineering methods and enables thus a functionalization of the granule surface. On Mixture of different fusion proteins is made simultaneously if necessary applied so that a multifunctionalization of the granule surface is created becomes.

Der N-Terminus (< Aminosäurerest 140 gemäß der Granulum-assoziierten Proteine PhaI und PhaF aus Pseudomonas oleovorans) der Proteine PhaF und PhaI aus Pseudomonaden vermittelt die Bindung der Proteine an den Polyester-Kern der Granula (siehe auch Prieto et al., 1999). Dieser N-terminale Abschnitt jeweils der Proteine PhaF und PhaJ wird durch gentechnische Methoden an Funktionsmoleküle fusioniert und ermöglicht somit eine Funktionalisierung der Granulum-Oberfläche. Ein Gemisch unterschiedlicher Fusionsproteine wird bei Bedarf gleichzeitig appliziert, so dass eine Multifunktionalisierung der Granulum-Oberfläche erzeugt wird.The N-terminus (<amino acid residue 140 according to the granule-associated Proteins PhaI and PhaF from Pseudomonas oleovorans) of the PhaF proteins and PhaI from Pseudomonads mediates the binding of the proteins the polyester core of the granules (see also Prieto et al., 1999). This N-terminal section of the PhaF and PhaJ proteins, respectively fused to functional molecules by genetic engineering methods and thus enables a functionalization of the granule surface. A mixture of different Fusion proteins are applied simultaneously if necessary, so that a multifunctionalization of the granule surface is generated.

Die oben angeführten Granulum-assoziierten Proteine werden ebenso durch Insertion von Epitopen gentechnisch modifiziert, um eine Antikörper-vermittelte Bindung zu erzielen. Grundsätzlich werden hier Peptid/Polypeptid-Insertionen durchgeführt, die eine spezifische Bindung vermitteln können (siehe auch Rehm und Hancock, 1996).The granule-associated listed above Proteins are also genetically engineered by inserting epitopes modified to be an antibody-mediated To achieve binding. Basically here peptide / polypeptide insertions are carried out that have a specific binding can mediate (see also Rehm and Hancock, 1996).

Kovalente Modifizierung von Aminosäureresten Granulum-assoziierter Proteine Granulum-assoziierte Proteine werden durch Aminosäure-spezifische chemische Reagenzien kovalent modifiziert werden. Durch diese Verknüpfung werden weitere Funktionsmoleküle wie z. B. Biotin an der Granulum-Oberfläche lokalisiert, die z. B. eine spezifische Bindung an andere Moleküle vermitteln (siehe auch Rehm et al., 1994).Covalent modification of amino acid residues Granule-associated proteins become granule-associated proteins by amino acid specific chemical reagents are covalently modified. Through this link other functional molecules such as B. localized biotin on the granule surface, the z. B. mediate a specific bond to other molecules (see also Rehm et al., 1994).

Kovalente Modifizierung von Molekülen an der Oberfläche des Polyester-Kerns Durch eine Vielzahl von Kopplungsreagenzien und/oder bifunktionellen Reagenzien werden die Moleküle, die sich an der Oberfläche des Polyester-Kerns befinden aktiviert und eine kovalente Verknüpfung mit Wirkstoffmolekülen wird durchgeführt.Covalent modification of molecules on the surface of the polyester core through a variety of coupling reagents and / or bifunctional reagents are the molecules that themselves on the surface of the polyester core are activated and a covalent link with drug molecules is carried out.

Literaturliterature

  • Hanley, S.Z., Pappin, D.J.C., Rahman, D., White, A.J., Elborough, K.M., Slabas, A.R. (1999) Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding. FEBS Letters 447: 99-105Hanley, S.Z., Pappin, D.J.C., Rahman, D., White, A.J., Elborough, K.M., Slabas, A.R. (1999) Re-evaluation of the primary structure of Ralstonia eutropha phasin and implications for polyhydroxyalkanoic acid granule binding. FEBS Letters 447: 99-105
  • Prieto MA, Buhler B, Jung K, Witholt B, Kessler B. (1999) PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. J Bacteriol. 181(3) : 858-68.Prieto MA, Buhler B, Jung K, Witholt B, Kessler B. (1999) PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. J Bacteriol. 181 (3): 858-68.
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Claims (20)

Ein Wirkstoffmolekül/Funktionsmolekül-Träger- und Lokalisierung-System bestehend aus biologisch abbaubaren Polyestern, die durch eine einfach oder mehrfach funktionalisierte Oberflächenschicht, die aus kovalent oder nicht kovalent an das Polyester gebundene Proteine und/oder aus nicht-kovalent an das Polyester gebundenen amphiphilen Molekülen besteht und die Herstellung der entsprechende PartikelAn active substance molecule / functional molecule carrier and Localization system consisting of biodegradable polyesters, by a single or multiple functionalized surface layer, the from proteins covalently or non-covalently bound to the polyester and / or from non-covalently bound to the polyester amphiphiles Molecules and the production of the corresponding particles Das System gemäß Patentanspruch 1 besteht aus einem Polyester-Kern, dessen Polyester-Zusammensetzung sämtliche Bausteine in unterschiedlichen Verhältnissen und Anordnungen umfasst, die von Polyestersynthetisierenden Enzymen zu einem Polyoxoester oder Polythioester polymerisiert werden könnenThe system according to claim 1 consists of a polyester core, the polyester composition all Includes building blocks in different ratios and arrangements, that of polyester synthesizing enzymes to a polyoxoester or polythioesters can be polymerized Das System gemäß Patentanspruch 2 liegt in Polyester-Nanopartikel (10-100 nm), -Submikropartikel (100-900 nm) und -Mikropartikel (1-3 μm) oder Filmen vor.The system according to claim 2 lies in polyester nanoparticles (10-100 nm), submicroparticles (100-900 nm) and microparticles (1-3 μm) or films. Die Partikel gemäß Patentanspruch 3 sind von Phospholipiden und/oder Etherlipiden und/oder Proteinen und/oder Lipopolysaccharide und/oder amphiphilen Molekülen, die an die Granulumoberfläche binden, umgeben und die Herstellung dieser OberflächenschichtThe particles according to claim 3 are of phospholipids and / or ether lipids and / or proteins and / or lipopolysaccharides and / or amphiphilic molecules which bind to the granule surface, surround and manufacture this surface layer Die Partikel gemäß Patentansprüche 2-4 werden in vivo in Eu- oder Archaebakterien gebildet und durch Wahl der Kultivierungsbedingungen (Vorstufensubstrate, sequentielle Vorstufensubstratfütterung), Mikroorganismen (Wildtypen, Mutanten und gentechnisch veränderte Mikroorganismen) und der Stoffwechselflusssteuerung (Inhibitoren von Stoffwechselflüssen, Etablierung von Stoffwechselwegen durch gentechnische Veränderung) wird die Zusammensetzung des Polyesters im Partikelkern gesteuertThe particles according to claims 2-4 are formed in vivo in Eu or Archaebacteria and by choice the cultivation conditions (pre-stage substrates, sequential pre-stage substrate feeding), Microorganisms (wild types, mutants and genetically modified microorganisms) and metabolic flow control (inhibitors of metabolic flows, establishment of metabolic pathways through genetic engineering) the composition controlled the polyester in the particle core Die Partikel gemäß Patentansprüche 2-5 werden bezüglich der Größe, durch Kontrolle des Verhältnisses von Polyester-synthesisierenden Enzymen zu polymerisierbaren Substrat und/oder durch die Kopienzahl von Phasinen, die PhaP aus Ralstonia eutropha oder Proteine mit PhaP-Funktion sind, gesteuertThe particles according to claims 2-5 be regarding the size, by Check the relationship from polyester-synthesizing enzymes to polymerizable substrates and / or by the copy number of phasins, the PhaP from Ralstonia eutropha or proteins with PhaP function are controlled Die Partikel gemäß Patentanspruch 4 sind von Proteinen umgeben, die Fusionen von Enzymen und/oder spezifische Bindung vermittelnden Proteinen mit PHA-Synthasen und/oder PHA-Depolymerasen und/oder Phasinen und/oder Proteinen mit Phasinfunktion und/oder PHA-Regulatorproteinen darstellenThe particles according to claim 4 are surrounded by proteins, the fusions of enzymes and / or specific binding mediating proteins with PHA synthases and / or PHA depolymerases and / or phasins and / or proteins with phasin function and / or represent PHA regulator proteins Die Partikel gemäß Patentansprüche 3 bis 6 werden durch Gelfiltrationschromatographie und/oder Dichtegradientenzentrifugation und/oder Ultrafiltration mit definierter Größe gewonnenThe particles according to claims 3 to 6 are by gel filtration chromatography and / or density gradient centrifugation and / or ultrafiltration with a defined size Die Oberfläche der Partikel gemäß Patentansprüche 4 bis 7 wird mit chemische Reagenzien aktiviert und Wirkstoffmoleküle werden über die aktivierten Stellen gebundenThe surface of the particles according to claims 4 to 7 is activated with chemical reagents and drug molecules are activated via the activated positions Die Partikel gemäß Patentanspruch 7 sind von Fusionproteinen umgeben, die aus einer an die Polyester-Oberfläche bindenden Domäne und einer spezifische Funktionen vermittelnden Domäne bestehenThe particles according to claim 7 are surrounded by fusion proteins, which consist of a binding to the polyester surface domain and a domain that provides specific functions Die Funktionen gemäß Patentanspruch 10 umfassen Enzymaktivitäten und/oder Obertlächenstrukturstabilisierung und/oder Vermittlung einer spezifischen molekularen InteraktionThe functions according to claim 10 include enzyme activities and / or surface structure stabilization and / or mediation of a specific molecular interaction Die Kopplungsreagenzien gemäß Patentanspruch 9 gehören folgenden Gruppen an: Bis-(2-oxo-3-oxazolydinyl)-Phosphonchlorid (BOP-C1), Bromotris-pyrrolidino-phosphoniumhexafluorophosphat (PyBroP), Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphat (PyBOP), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphat (HBTU), Dicyclohexyl-carbodümide, Disuccinimidyl carbonate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), Bis-(2-oxo-3-oxazolydinyl)-phosphin, Diisopropyl-carbodiimide (DIPC), 2-(1N-benzotrioxazolyl)-1,1,3,3-tetramethyluroniumtetrafluoroborat (TBTU), 2-(5-norboren-2,3-dicarboxyimido)-1,1,3,3-tetramethyluroniumtetrafluoroborat (TNTU), para-Nirophenylchloroformate, and O-(N-succinimidy)-1,1,3,3- tetramethyluronium tetrafluoroborate (TSTU), Aminosäurerest modifizierende ReagenzienThe coupling reagents according to claim 9 belong the following groups: bis- (2-oxo-3-oxazolydinyl) phosphonchloride (BOP-C1), Bromotris pyrrolidinophosphonium hexafluorophosphate (PyBroP), Benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP), 2- (1H-benzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), dicyclohexyl carbodiimide, Disuccinimidyl carbonate, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC), bis (2-oxo-3-oxazolydinyl) phosphine, diisopropyl carbodiimide (DIPC), 2- (1N-benzotrioxazolyl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2- (5-norborene-2,3-dicarboxyimido) -1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), para-Nirophenylchloroformate, and O- (N-succinimidy) -1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), amino acid residue modifying reagents Zur Aktivierung der Partikeloberfläche gemäß Patentanspruch 9 werden folgende Reagenzien eingesetzt: Triethylamine, N-Methylmorpholin, Pyridine, 1,8-Diazabicyclo-[5,4,0]-7undecen, N, N-Dimethylaminopyridine und N, N-DüsopropylethylaminTo activate the particle surface according to claim 9, the following are Reagents used: triethylamine, N-methylmorpholine, pyridines, 1,8-diazabicyclo- [5,4,0] -7undecene, N, N-dimethylaminopyridines and N, N-diisopropylethylamine Zur Aktivierung der Partikeloberfläche gemäß Patentanspruch 9 werden folgende Additive eingesetzt: Hydroxybenzotriazole, Pentafluorophenol und N-Hydroxy-5-norboren-endo-2,3-dicarboximid.To activate the particle surface according to claim 9, the following are Additives used: hydroxybenzotriazoles, pentafluorophenol and N-hydroxy-5-norboren-endo-2,3-dicarboximide. Die Wirkstoffe gemäß Patentanspruch 1 oder 9 umfassen Anti-Tumor-Agenzien wie z. B. Dideoxyinosin, Floxuridin, 6-Mercaptopurin, Doxorubicin, Daunorubicin, 1-Darubicin, Cisplatin, Methotrexat, TaxoI etc.; Antibiotika wie z.B. Erythromycin, Vancomycin, Oleandomycin, Ampicillin, etc.; Anticoaguranzien wie z.B. Heparin; Germizide wie z.B. ara-A, Acrylguanosin, Nordeoxyguanosin, Azidothymidin, Dideoxyadenosin, Dideoxythymidin, etc.; Antiarrythmische Agenzien; und Wirkstoftvorstufen und -derivate der angeführten WirkstoffgruppenThe active ingredients according to claim 1 or 9 include anti-tumor agents such as. B. dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, 1-darubicin, cisplatin, methotrexate, TaxoI, etc .; Antibiotics such as erythromycin, Vanco mycin, oleandomycin, ampicillin, etc .; Anticoagurants such as heparin; Germicides such as ara-A, acrylic guanosine, north oxy guanosine, azidothymidine, dideoxy adenosine, dideoxy thymidine, etc .; Antiarrhythmic agents; and drug precursors and derivatives of the listed drug groups Die Wirkstoffe gemäß Patentanspruch 1 oder 9 oder Funktionen gemäß Patentanspruch 11 sind Moleküle von biologisch aktiven Verbindungen und umfassen Peptide, Proteine, Therapeutika, Diagnostika, und nicht-biologische Moleküle wie z.B. Pestizide, Herbizide und DüngemittelThe active ingredients according to claim 1 or 9 or functions according to claim 11 are molecules of biologically active compounds and include peptides, proteins, Therapeutics, diagnostics, and non-biological molecules such as Pesticides, herbicides and fertilizers Peptide gemäß Patentanspruch 16 werden aus einer Gruppe ausgewählt, die Insulin, Calcitonin, ACTH, Glucagon, Somatostatin, Somatotropin, Somatomedin, Parathyroidhormon, Erythropoietin, hypothalamische Freisetzungfaktoren, Prolactin, Thyroid-stimulierendes Hormon, Endorphine, Enkephaline, Vasopressine, nicht natürlich vorkommende Opiate, Superoxiddismutase, Interferon, Asparaginase, Arginase, Arginindeaminase, Adenosindeaminase, Ribonuclease, Trypsin, Chymotrypsin und PepsinPeptides according to claim 16 are selected from a group consisting of insulin, calcitonin, ACTH, Glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, Erythropoietin, hypothalamic release factors, prolactin, Thyroid stimulating hormone, endorphins, enkephalins, vasopressins, not natural occurring opiates, superoxide dismutase, interferon, asparaginase, Arginase, arginine deaminase, adenosine deaminase, ribonuclease, trypsin, Chymotrypsin and pepsin Herstellungsverfahren für Partikel gemäß Patentanspruch 1 in dem Patentansprüche 2 bis 17 kombiniert oder unabhängig von einander berücksichtigt werdenManufacturing process for Particles according to claim 1 in the claims 2 to 17 combined or independent taken into account by each other become Pharmazeutische Zubereitungen, die als Wirkstoff die Partikel gemäß Patentansprüche 1 bis 18 enthaltenPharmaceutical preparations containing the particles as an active ingredient according to claims 1 to 18 contain Pharmazeutische Zubereitungen gemäß Patentansprüche 15 und 19, die eine immunomodulatorische, antiinfektiöse und/oder antitumorale therapeutische Wirkung aufweisenPharmaceutical preparations according to claims 15 and 19, which is an immunomodulatory, anti-infectious and / or anti-tumor therapeutic Have effect
DE10240035A 2002-08-30 2002-08-30 Biogenic polyester particles of a defined size with functionalized surfaces: manufacturing processes and pharmaceutical preparations containing them Withdrawn DE10240035A1 (en)

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DE10240035A DE10240035A1 (en) 2002-08-30 2002-08-30 Biogenic polyester particles of a defined size with functionalized surfaces: manufacturing processes and pharmaceutical preparations containing them
NZ569097A NZ569097A (en) 2002-08-30 2003-08-22 Method for providing a rapidly usable low-cost transport system for biologically active substances that permits effective and reliable transport of active ingredients in the animal organism
SG200701490-5A SG153663A1 (en) 2002-08-30 2003-08-22 Process for the production of biodegradable, functionalised polymer particles and use thereof as pharmaceutical supports
EP10181200A EP2302064A1 (en) 2002-08-30 2003-08-22 Method for producing biodegradable, functionalised polymer particles, and use of the same as medicament carriers
DK03747816.1T DK1539976T3 (en) 2002-08-30 2003-08-22 Process for the preparation of biodegradable, functionalized polymer particles and their use as drug carriers
DE50313457T DE50313457D1 (en) 2002-08-30 2003-08-22 METHOD FOR THE PRODUCTION OF BIODEGRADABLE, FUNCTIONALIZED POLYMER PARTICLES AND THEIR USE AS MEDICAMENTAL CARRIER
AT03747816T ATE497543T1 (en) 2002-08-30 2003-08-22 METHOD FOR PRODUCING BIODEGRADABLE, FUNCTIONALIZED POLYMER PARTICLES AND THEIR USE AS DRUG CARRIERS
AU2003266922A AU2003266922B2 (en) 2002-08-30 2003-08-22 Method for producing biodegradable, functionalised polymer particles, and use of the same as medicament carriers
PCT/DE2003/002799 WO2004020623A2 (en) 2002-08-30 2003-08-22 Method for producing biodegradable, functionalised polymer particles, and use of the same as medicament carriers
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JP2004531694A JP5160729B2 (en) 2002-08-30 2003-08-22 Method for producing functionalized biodegradable polymer particles and pharmaceutical carrier using the polymer particles
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ES03747816T ES2360617T3 (en) 2002-08-30 2003-08-22 PROCEDURE FOR THE PREPARATION OF FUNCTIONALIZED AND BIODEGRADABLE POLYMER PARTICLES AND FOR USE AS VEHICLES FOR MEDICINES.
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US10/525,955 US7622277B2 (en) 2002-08-30 2003-08-22 Process for the production of biodegradable, functionalised polymer particles, and use thereof as pharmaceutical supports
AU2009210395A AU2009210395A1 (en) 2002-08-30 2009-08-20 Method for producing biodegradable, functionalised polymer particles, and use of the same as medicament carriers
US12/588,227 US20100086972A1 (en) 2002-08-30 2009-10-08 Process for the production of biodegradable, functionalised polymer particles and use thereof as pharmaceutical supports
CY20111100420T CY1113685T1 (en) 2002-08-30 2011-04-29 METHOD OF PRODUCTION OF BIOLOGICALLY DEVELOPABLE, FUNCTIONAL POLYMERIC PARTICLES AND USE OF THESE CARE MEDICINES
JP2012152732A JP2012249634A (en) 2002-08-30 2012-07-06 Method for producing biodegradable functionalized polymer particle, and use of the same as medicament carrier
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